A reversible, cellular cholesterol lipidosis was studied to probe the relationship between the exit of cholesterol from lysosomes and the functional integrity of the Golgi. Although this progesterone-induced accumulation of cholesterol in lysosomes during LDL-uptake is extensive, it did not block enrichment of membranes of trans Golgi cisternae with cholesterol. Thus, progesterone and the Niemann Pick Type-C (NP-C) mutation both induce an accumulation of cholesterol in lysosomes which is linked to a more distal block in cholesterol trafficking at the trans Golgi cisternae. Endoplasmic reticulum is an additional intracellular site at which the processing and transport of cholesterol can be disrupted. During uptake of LDL, inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) with 58035 induced an extensive accumulation of unesterified cholesterol in rough endoplasmic reticulum. There was no visible sequestration of cholesterol in lysosomes even though the amount of unesterified cholesterol in cells treated with the ACAT inhibitor reached levels seen in both NP-C and progesterone-treated cells. Although 58035 blocks ACAT, the drug enhances other homeostatic responses whereas, the progesterone induced block and NP-C mutation inhibit all homeostatic responses. These differences in potency of cholesterol regulation of cell homeostasis can now be related to organelle site of cholesterol accumulation. Cholesterol transfer to endoplasmic reticulum may be necessary to elicit these cellular responses. A similar lipase hydrolyses lipid stores in adipocytes and steroidogenic cells (hormone sensitive lipase, HSL). Immunocytochemical studies, showed HSL was present on the surface monolayer of most triacylglycerol containing lipid droplets in adipocytes stimulated with isoproterenol, whereas little HSL was associated with the surface of lipid droplets in unstimulated adipocytes. Thus, hormonal stimulation of adipocytes increases access of intracellular HSL to its triacylglycerol substrate. A second pool of HSL was found in mitochondria of both adipocytes and steroidogenic cells. The location of HSL in mitochondria and their close relationship with the surface of intracellular lipid droplets would facilitate transfer of lipolytic products, cholesterol and fatty acids, to mitochondria for further metabolism.